G1/S restriction point coordinates phasic gene expression and cell differentiation

Pluripotent embryonic stem cells have a unique cell cycle structure with a suppressed G1/S restriction point and little differential expression across the cell cycle phases. Here, we evaluate the link between G1/S restriction point activation, phasic gene expression, and cellular differentiation. Expression analysis reveals a gain in phasic gene expression across lineages between embryonic days E7.5 and E9.5. Genetic manipulation of the G1/S restriction point regulators miR-302 and P27 respectively accelerates or delays the onset of phasic gene expression in mouse embryos. Loss of miR-302-mediated p21 or p27 suppression expedites embryonic stem cell differentiation, while a constitutive Cyclin E mutant blocks it. Together, these findings uncover a causal relationship between emergence of the G1/S restriction point with a gain in phasic gene expression and cellular differentiation.


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All studies must disclose on these points even when the disclosure is negative.  5 and S1 and S10 have associated raw data available via GSE142215. There are no restrictions on availability of the data.
Sample sizes are indicated in the figure legends. Sample sizes were based on previous experiments or pilot experiments. The sizes were chosen to enable comparisons of interest, for example between samples with different genotypes, at different stages or from different lineages. The metrics used to evaluate reproducibility and make statistical comparisons between the groups are detailed in the figure captions and methods of the manuscript.
All data were included with the exception of outliers in the 3' end count libraries of phase-sorted embryos between E7.5 and E9.5 We used global UMAPs to quality control of the mRNA-seq samples, where the expectation is that samples of the same embryonic stage would cluster together. The samples that did not cluster by stage were removed. Inspection of the expression profiles suggests that the failure of these samples to cluster with samples of the same group was likely due to contaminating yolk sac or poor overall library quality.
We also excluded a comparison of phasic expression between wt and ccne1sd mutant cells during neural differentiation because the wt cells had differentiated to the point where the data was heavily confounded by expression of genes from postmitotic cells All attempts at replication were successful.
Distinct embryos were used for replicates in all cases except the single-cell RNA-seq knockout experiments ( Figure 4) where only a single capture was performed for each of mir-302-/-and p27-/-. Notably, the single-cell RNA-seq experiments on knockout embryos were a complimentary approach to the bulk RNA-seq on the same mutants. In the single-cell experiments, cellular permutations were used as internal replicates for the experiments (see methods).
Aside from the single-cell experiments on mutant embryos, each experiment was replicated at least twice. The exact number of replications for each experiment is provided in the associated caption.
Groups were designated based on embryonic stages (Downs and Davies, 1993), cell cycle phases (gating of Hoechst staining), lineages (defined by expression) and genotypes. Additional detail on each grouping is provided in the manuscript.
The majority of studies were not blinded as the samples were primarily handled by an individual experimentalist. Quantification of the overlap in Cyclin E1 and Cyclin B1 protein in E7.5, E8.5 and E9.5 was performed by an individual blinded to the remainder of this study.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. GFP: GFP was evaluated in mir-302 GFP-knock-in reporter animals relative to wt embryos. The observed distribution is consistent with the distribution evident in fresh samples. Please see the manufacturer's website for additional validation and associated citations: https://www.aveslabs.com/products/anti-green-fluorescent-protein-antibody-gfp OCT4/NANOG: Both were evaluated in mouse embryonic stem cells for positive staining, and compared to mouse embryonic fibroblast and neuroepithelium as negative controls. Please see the manufacturer's website for additional validation and associated citations: https://www.bdbiosciences.com/en-us/products/reagents/microscopy-imaging-reagents/immunofluorescence-reagents/ purified-mouse-anti-oct3-4.611202 (OCT4), and https://www.abcam.com/nanog-antibody-ab80892.html (NANOG).

Materials & experimental systems n/a Involved in the study
pRb: Evaluated by flow in mouse embryonic stem cells using an Rb knockout line as a control. Also as expected, pRb also increases following G1/S transit. Please see the manufacturer's website for additional validation and associated citations: https:// www.cellsignal.com/products/antibody-conjugates/phospho-rb-ser807-811-d20b12-xp-rabbit-mab-pe-conjugate/11917 TUJ1: Used embryonic sections with postmitotic neurons as well as in vitro differentiated neurons as positive controls. ES cells and non-neuronal embryonic regions served as negative controls. Please see the manufacturer's website for additional validation and associated citations: https://www.abcam.com/beta-iii-tubulin-antibody-neuronal-marker-ab18207.html Cyclin E1: Compared positive signal in mitotic cells to negative signal in post-mitotic cells. Please see the manufacturer's website for additional validation and associated citations: https://www.rndsystems.com/products/human-cyclin-e1-antibody_af6810 (IF), and https://www.cellsignal.com/products/primary-antibodies/cyclin-e1-d7t3u-rabbit-mab/20808 (Flow) Cyclin B1: Evaluated cellular distribution (nuclear in M phase and cytoplasmic in other cell cycle phases). Please see the manufacturer's website for additional validation and associated citations: https://www.cellsignal.com/products/primary-antibodies/ cyclin-b1-antibody/4138 V6.5 embryonic stem (ES) cells (Jaenisch lab); DGCR8-/-ES cells (derived in house in the Blelloch lab); SBR reporter ES cells (Suter lab). NIH/3T3 fibroblasts (ATCC). Novel mutants generated as described in methods.
The DGCR8-/-ES cells were generated in the Blelloch lab and genotyped by southern and western blots.
The SBR ESCs were generated in the Suter lab, and authenticated by genotyping (PCR) and using directed differentiations.
The 3T3 cells were authenticated via morphology.